A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

Suin, Vanessa; Naze, Florence; Francart, Aurélie; Lamoral, Sophie; Craeye, Stéphane De; Kalai, Michaël; Gucht, Steven Van

doi:10.1155/2014/256175

Cited by 15 publications

(16 citation statements)

References 44 publications

(54 reference statements)

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“…The viral RNA load was determined using real-time reverse transcriptase polymerase chain reaction (RT-qPCR), as described by Suin et al [36] , in the whole brain, or in the olfactory bulbs, mid (cerebrum and diencephalon) and anterior (hindbrain and cerebellum) parts of the brain. Primers recognize the nucleocapsid region of genomic RNA [36] . Previously, Suin et al [36] found a good correlation between viral RNA load and infectious virus titer in the brain.…”

Section: Methodsmentioning

confidence: 99%

“…Primers recognize the nucleocapsid region of genomic RNA [36] . Previously, Suin et al [36] found a good correlation between viral RNA load and infectious virus titer in the brain. Brain samples were homogenised using a tissue homogenizer (Bullet Blender, Next Advance, New York, USA) and 5 mm stainless steel beads in 350–1000 µl lysis buffer (RLT buffer, as supplied with Qiagen RNeasy kit, with 1% β-mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

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Protective Effect of Different Anti-Rabies Virus VHH Constructs against Rabies Disease in Mice

et al. 2014

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Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Protective Effect of Different Anti-Rabies Virus VHH Constructs against Rabies Disease in Mice

et al. 2014

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“…Furthermore, compared to the hemi-nested RT-PCR, the established RT-qPCRs offer some important advantages in reduction of workload and run time, while providing a higher sensitivity compared to the conventional RT-PCR. Finally our assays, particularly the L-gene based assay, could be a complement for existing methods for rabies diagnosis (21,31,46,48,49) with a high specificity, sensitivity and repeatability and more suitable for broad detection of African RABV strains.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

Faye

Dacheux

Weidmann

et al. 2017

Journal of Virological Methods

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Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy.

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“…Real-time RT-PCR based assays have improved sensitivity and specificity compared to other methods, highlighting the potential of a real-time RT-PCR assay to become a leading assay for rabies laboratory diagnosis. Over the last decade many studies have been published assessing real-time RT-PCR assays for the detection of RABV and other lyssaviruses via SYBR Green or TaqMan probe [17–21] methods. TaqMan probe based real-time RT-PCR assays often have lower sensitivity relative to SYBR Green based assays due to the sensitivity of TaqMan probes to the diversity of target sequences in the genomes of different RABV and other lyssaviruses [18, 22–24].…”

Section: Introductionmentioning

confidence: 99%

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

et al. 2017

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Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

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A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

Cited by 15 publications

References 44 publications

Protective Effect of Different Anti-Rabies Virus VHH Constructs against Rabies Disease in Mice

Protective Effect of Different Anti-Rabies Virus VHH Constructs against Rabies Disease in Mice

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

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